Diffuse intrinsic pontine glioma (DIPG) is the most deadly solid tumor in children, with rapid disease progression, poor response to therapy, and average survival less than one year from diagnosis. Epigenetic regulation of gene expression has been implicated in a variety of human diseases, including cancer. Recently, point mutations in histone H3 have been identified in up to 80% of DIPG, causing altered chromatin function and extensive transcriptome reprogramming. Here, we describe a hypothesis-driven research plan to elucidate the mechanism by which histone H3 post-translational modifications contribute to pediatric brainstem glioma (DIPG) formation and progression. A career development plan is also described to facilitate successful transition from a mentored investigator to an independent investigator. In support of the hypothesis that histone modifications in H3K27M mutant pediatric brainstem glioma contribute to disease pathogenesis, we will characterize alterations in gene expression in DIPG associated with specific histone H3 transcriptional regulatory marks. Further, we will investigate the enzymatic mechanisms responsible for these histone modifications, to determine if these represent rational therapeutic targets. In order to investigate the effect of Histone H3 modifications on gene transcription in DIPG, we will perform chromatin immunoprecipitation in association with DNA sequencing (ChIP-Seq) to characterize genomic locations of specific H3 marks, and correlate these findings with gene expression (RNA-Seq). We will then characterize the effects of altered expression of specific oncogenes (PDGFRA) and tumor suppressor genes (p21), and enzymes responsible for identified H3 modifications, on DIPG tumor biology in vitro and in vivo. Human H3K27M mutant and wild type DIPG tumor cells will be used for in vitro studies, while a mouse xenograft model of DIPG will be used for in vivo investigations. The aims of this proposal are: 1) To determine DIPG transciptomes with respect to histone H3 modifications; 2) To elucidate the enzymatic basis of histone H3 modifications in DIPG, and determine whether specific enzymatic activities are actionable for treating DIPG; and 3) To validate specific oncogene activations and tumor suppressor gene inactivations as causally associated with DIPG malignant phenotypes The findings of this proposal will demonstrate the effect of epigenetic dysregulation on gene expression in pediatric brainstem glioma, providing novel insight on the mechanism of DIPG tumorigenesis and a potential for developing rational, effective treatment for children with this disease.